J. L. Ebersole

Omega-3 Fatty Acid Effect on Alveolar Bone Loss in Rats

Gingival inflammation and alveolar bone resorption are hallmarks of adult periodontitis, elicited in response to oral micro-organisms such as Porphyromonas gingivalis. We hypothesized that omega (ω)-3 fatty acids (FA) dietary supplementation would modulate inflammatory reactions leading to periodontal disease in infected rats. Rats were fed fish oil (ω-3 FA) or corn oil (n-6 FA) diets for 22 weeks and were infected with P. gingivalis. Rats on the ω-3 FA diet exhibited elevated serum levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), documenting diet-induced changes. PCR analyses demonstrated that rats were orally colonized by P. gingivalis; increased IgG antibody levels substantiated this infection. P. gingivalis-infected rats treated with ω-3 FA had significantly less alveolar bone resorption. These results demonstrated the effectiveness of an ω-3 FA-supplemented diet in modulating alveolar bone resorption following P. gingivalis infection, and supported that ω-3 FA may be a useful adjunct in the treatment of periodontal disease. Abbreviations: PUFA, polyunsaturated fatty acid; EPA, eicosapentanoic acid; DHA, docosahexanoic acid; and PCR, polymerase chain-reaction.

Role of Glutathione Metabolism of Treponema denticola in Bacterial Growth and Virulence Expression

ABSTRACT Hydrogen sulfide (H2S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis. Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H2S. The metabolic pathway leading to H2S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for T. denticola to produce H2S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H2S production by this microorganism. In addition to H2S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H2S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in T. denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56°C) and freezing (−20°C), by sonication, and by exposure to Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T. denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.

Caloric restriction and chronic inflammatory diseases

A reduction in calorie intake [caloric restriction (CR)] appears to consistently decrease the biological rate of aging in a variety of organisms as well as protect against age-associated diseases including chronic inflammatory disorders such as cardiovascular disease and diabetes. Although the mechanisms behind this observation are not fully understood, identification of the main metabolic pathways affected by CR has generated interest in finding molecular targets that could be modulated by CR mimetics. This review describes the general concepts of CR and CR mimetics as well as discusses evidence related to their effects on inflammation and chronic inflammatory disorders. Additionally, emerging evidence related to the effects of CR on periodontal disease in non-human primates is presented. While the implementation of this type of dietary intervention appears to be challenging in our modern society where obesity is a major public health problem, CR mimetics could offer a promising alternative to control and perhaps prevent several chronic inflammatory disorders including periodontal disease.

Extracorporeal Shock Wave Therapy Induces Alveolar Bone Regeneration

Periodontal inflammation with alveolar bone resorption is a hallmark of periodontitis. We hypothesized that extracorporeal shock wave therapy (ESWT) could promote the regeneration of alveolar bone following Porphyromonas gingivalis-induced periodontitis in rats. Rats were infected with P. gingivalis for 10 wks, which caused alveolar bone resorption. The rats were then treated with a single episode of 100, 300, or 1000 impulses of shock wave on both cheeks at energy levels 0.1 mJ/mm2. Alveolar bone levels were determined at 0, 3, 6, and 12 wks following ESWT and compared with those in untreated controls. Infected rats treated with 300 and 1000 impulses demonstrated significantly improved alveolar bone levels at 3 wks compared with untreated controls, and the improved levels remained for at least 6 wks in most rats. The results demonstrated effective regeneration of alveolar bone by ESWT and suggested that ESWT should be evaluated as an adjunct in the regeneration of periodontal tissues following periodontal disease. Abbreviations: ESWT, extracorporeal shock wave therapy; PCR, polymerase chain-reaction.

Differential gender effects of a reduced-calorie diet on systemic inflammatory and immune parameters in nonhuman primates

BACKGROUND AND OBJECTIVE Dietary manipulation, including caloric restriction, has been shown to impact host response capabilities significantly, particularly in association with aging. This investigation compared systemic inflammatory and immune-response molecules in rhesus monkeys (Macaca mulatta). MATERIAL AND METHODS Monkeys on continuous long-term calorie-restricted diets and a matched group of animals on a control ad libitum diet, were examined for systemic response profiles including the effects of both gender and aging. RESULTS The results demonstrated that haptoglobin and alpha1-antiglycoprotein levels were elevated in the serum of male monkeys. Serum IgG responses to Campylobacter rectus, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were significantly elevated in female monkeys. While only the antibody to Fusobacterium nucleatum was significantly affected by the calorie-restricted diet in female monkeys, antibody levels to Prevotella intermedia, C. rectus and Treponema denticola demonstrated a similar trend. CONCLUSION In this investigation, only certain serum antibody levels were influenced by the age of male animals, which was seemingly related to increasing clinical disease in this gender. More generally, analytes were modulated by gender and/or diet in this oral model system of mucosal microbial challenge.

Antigen Specificity of Serum Antibody in A. actinomycetemcomitans-infected Periodontitis Patients:

We hypothesized that serum antibody with selected antigen specificities would relate to infection and disease in the patients and, thus, describe the characteristics of potential protective antibody. This study used serum samples from 24 periodontitis patients with subgingival infection and elevated serum IgG antibody to A. actinomycetemcomitans to define the antigenic specificities of IgG, IgM, IgA, and IgGl-4 antibody to A. actinomycetemcomitans strain Y4 outer membrane antigens (OMA). Uniform IgG antibody (> 70% of the patients) was noted to antigens with Mr of 65, 38, 29, and 17 kDa. Both IgA and IgM specificities reflected those shown for IgG in each patient. IgGl and IgG2 antibody reacted with several OMA bands in each patient, while IgG3 antibodies were directed to numerous OMA bands in many patients and represented the most broad-based response. The IgG4 response patterns were limited to a few OMA bands. We noted a prominent occurrence of IgG reactions with OMA bands that were characteristic for individual patients. The frequency of responses to OMA of higher Mr (i.e., > 80 kDa) and to the 34-, 31-, and 24-kDa antigens was positively related to the total IgG antibody levels. Antibody reactive with OMA bands at 65-, 38-, 29-, 17-, 15-, and 11-kDa antigens was detected in patients with few to many teeth infected with A. actinomycetemcomitans. Furthermore, patients with a high percentage of teeth with ≥ 6 mm pockets had a decreased frequency of responses to the high-Mr antigens (i.e. > 90 kDa) as well as to the 58-kDa antigen. These findings indicate that human antibody reactivities with specific OMA bands of A. actinomycetemcomitans: (i) are positively correlated with the level of serum antibody, (ii) are associated with the number of teeth infected, and (iii) describe differences in the severity of the disease as measured by the frequency of teeth with deep pockets.

Utility of Salivary Biomarkers for Demonstrating Acute Myocardial Infarction

The comparative utility of serum and saliva as diagnostic fluids for identifying biomarkers of acute myocardial infarction (AMI) was investigated. The goal was to determine if salivary biomarkers could facilitate a screening diagnosis of AMI, especially in cases of non-ST elevation MI (NSTEMI), since these cases are not readily identified by electrocardiogram (ECG). Serum and unstimulated whole saliva (UWS) collected from 92 AMI patients within 48 hours of chest pain onset and 105 asymptomatic healthy control individuals were assayed for 13 proteins relevant to cardiovascular disease, by Beadlyte technology (Luminex®) and enzyme immunoassays. Data were analyzed with concentration cut-points, ECG findings, logistic regression (LR) (adjusted for matching for age, gender, race, smoking, number of teeth, and oral health status), and classification and regression tree (CART) analysis. A sensitivity analysis was conducted by repetition of the CART analysis in 58 cases and 58 controls, each matched by age and gender. Serum biomarkers demonstrated AMI sensitivity and specificity superior to that of saliva, as determined by LR and CART. The predominant discriminators in serum by LR were troponin I (TnI), B-type natriuretic peptide (BNP), and creatine kinase-MB (CK-MB), and TnI and BNP by CART. In saliva, LR identified C-reactive protein (CRP) as the biomarker most predictive of AMI. A combination of smoking tobacco, UWS CRP, CK-MB, sCD40 ligand, gender, and number of teeth identified AMI in the CART decision trees. When ECG findings, salivary biomarkers, and confounders were included, AMI was predicted with 80.0% sensitivity and 100% specificity. These analyses support the potential utility of salivary biomarker measurements used with ECG for the identification of AMI. Thus, saliva-based tests may provide additional diagnostic screening information in the clinical course for patients suspected of having an AMI.

Oral Epithelial Cell Responses to Multispecies Microbial Biofilms

This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms.

Effects of Low-energy Shock Waves on Oral Bacteria

We have recently demonstrated that extracorporeal shock-wave therapy (ESWT) is effective in promoting the healing of dermal wounds and in regenerating alveolar bone lost through periodontal disease. The objective of the present study was to determine any antibacterial effect of ESWT on oral bacteria. Monoculture suspensions of 6 bacterial species were treated with 100 to 500 pulses of ESWT at energy flux densities (EFD) of 0.12 mJ/mm2, 0.22 mJ/mm2, and 0.3 mJ/mm2. Following treatment, aliquots were plated for viability determination and compared with untreated controls. ESWT showed a significant microbicidal effect for Streptococcus mutans and an unencapsulated strain of Porphyromonas gingivalis following as few as 100 pulses at 0.3 mJ/mm2 (p ≤ 0.001). In addition, a significant disruption of bacterial aggregates was observed at lower EFDs. No significant reduction in viability was observed for all other bacteria at EFDs and pulses tested (p > 0.05). These findings suggest that low-energy ESWT may be bactericidal for selected oral bacteria.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature‐induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid‐pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E2 (PGE2) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)‐6 occurred in the experimental animals by the time of delivery. IL‐8, MCP‐1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE2, LBP, IL‐8 and MCP‐1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL‐6, IL‐8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE2, macrophage chemotactic protein (MCP)‐1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature‐induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals.